The Notch pathway is a conserved signaling system used extensively throughout embryonic development that continues to function in the maintenance of tissues and stem cells in adults. Given the large repertoire of cellular processes dependent on Notch, it is not surprising that both gains and losses in signaling have been linked to developmental syndromes, stroke, Alzheimers disease and cancer. The identification of Notch as a potential therapeutic target, along with its importance in manipulating embryonic and adult stem cells, underscore the need to clearly understand the mechanisms regulating this key signaling system. Ligands that bind and activate Notch are integral membrane proteins and signaling is dependent on direct cell-to-cell contact. Notch signaling induced by ligand involves a series of proteolytic cleavage events to release the Notch intracellular domain that functions as a transcription factor, thereby allowing the Notch receptor to function directly in signal transduction. Notch ligands and activating proteases have been identified, however, it is still unclear how interactions between Notch ligand and receptor cells activate signaling. Although it is clear that endocytosis by the ligand cell is critical for activation of signaling in the Notch cell, the mechanisms whereby endocytosis triggers signaling have remained elusive. The experiments proposed here will address a major obstacle to obtaining a clear understanding of Notch signaling mechanisms, since they will discriminate as to whether ligand endocytosis is required for ligand recycling prior to interactions with Notch, or whether in fact ligand endocytosis is required following binding to Notch to effect proteolytic activation for downstream signaling. This application outlines a research plan that combines molecular, biochemical and cellular approaches using mammalian cell culture to obtain a comprehensive understanding of the molecular machinery and endocytic mechanism used by ligand cells to activate signaling. Our preliminary studies indicate that ligand cells use an epsin-dependent mode of clathrin-mediated endocytosis to promote proteolytic activation of Notch. We now propose to obtain a complete molecular description of the clathrin-coated endocytic structure to gain mechanistic insight into how ligand endocytosis functions in Notch activation. Epsin has been implicated in recycling to produce an active ligand prior to interaction with Notch. We will directly test this hypothesis by determining first whether epsin is required for ligands to recycle and second, whether ligand recycling is required for signaling activity. Given that epsins recruit ubiquitinated endocytic cargo to clathrin- coated pits and Notch ligands must be ubiquitinated to activate signaling, we will determine if ligand ubiquitination is critical for interactions with epsin and the formation of a functionally distinct clathrin-coated endocytic structure, and whether interactions between ligand and Notch cells stimulate ligand ubiquitination. Our hypothesis that Notch is activated via a molecularly distinct form of clathrin-mediated ligand endocytosis represents a fundamentally new model for endocytic activation of a signaling receptor.